To tackle the increasing rate of marsupial extinction in Australia, a deep understanding of their reproductive biology is required to develop appropriate assisted reproduction technologies. The uterine environment is of central importance to marsupial reproduction given the extended preimplantation phase of embryo development during which the embryo is reliant on uterine secretions. Our recent work has characterized the uterine secretions across gestation, however there is a paucity of models with which to examine such marsupial maternal-fetal communications in vitro. Thus, this research aimed to develop an immortalised cell line representative of pertinent cell populations within the marsupial uterus appropriate for examination of the molecular profile of the uterine fluid.
Endometrial cells from the fat-tailed dunnart (Sminthopsis crassicaudata) were isolated and immortalised using SV-40 T-antigen technology. Cells were exposed to hormones representative of the gestational period (β-estradiol and progesterone) and assessed for: karyotype, proliferation (MTS assay), and gene expression (RNA-seq and qPCR).
A heterogenous population of cells was immortalised and named dunnart mixed endometrial cells 2 (dMEC2). These cells have a combination of fibroblastic and polygonal morphologies, with a polyploid karyotype. They express appropriate hormone receptors (Esr1, Pgr, Ar), and application of exogenous hormones did not impact cell morphology or proliferation (P ≥ 0.16 at each time point) but influenced gene expression profiles (RNA-seq). qPCR confirms the hormonal control of histotrophic components (as previously determined by our multi-omic analysis of uterine fluid) within this cell line.
dMEC2 is a powerful new research tool and has been used to identify hormonally responsive pathways in the marsupial uterus. Indeed, expression and regulation of uterine fluid related transcripts demonstrate the value of dMEC2 for marsupial reproductive biology. By developing an endometrial cell line, we expand the tool-box for marsupial researchers and provide new avenues for marsupial uterine research.